Regulatory Innovations Driving the Evolution of the Mammalian Neocortex. A major question in biology is how complex phenotypes originate and evolve over time. Regulatory innovations are thought to contribute to major morphological transitions, but the mechanisms underlying these innovations have not been extensively studied. The research outlined in this proposal aims to study the role of regulatory change in morphological evolution, using the mammalian neocortex as a test case. While the midbrain and hindbrain of higher vertebrates is conserved, the forebrain is a structure that has undergone dramatic changes during vertebrate and mammalian evolution. Mammals are unique in having a six-layered cortex; thus, the evolution of the mammalian neocortex is thought to have been a major transition. I hypothesize that this transition was driven by the large-scale co-option of genes into a cortical developmental program. This process may have involved widespread exaptation of repeat elements, the generation of new regulatory sequence by other means, or mammal-specific functional changes to ancient regulatory elements. It is known that mammals share a vast number of repeat element exaptations, although the function of these exapted elements in mammalian development is unknown. In this research I will test the hypothesis that the origin of the neocortex involved widespread exaptation of novel mammalian sequences such as transposable elements and changes in the function of ancient elements. First, I will use ChIP-seq to generate regulatory maps of the embryonic pallium of human, mouse, and chicken as the outgroup species. The mammalian neocortex is derived from the pallium, which is homologous in the species being tested. I will compare the distribution and brain-specific utilization of cis-regulatory elements in these species using cohesin, H3K4me2, and H3K27ac, as preliminary work in our lab has shown that these marks capture about 70% of all experimentally validated developmental enhancers. I will identify and characterize regulatory innovations specific to the developing mammalian cortex. I will determine the fraction of regulatory innovations derived from novel and ancient sequences, and further investigate the origin and characteristics of the novel sequences. I will also compare whole transcriptome profiles of human, mouse, and chicken embryonic pallium. The transcriptome data will allow me to identify genes co-opted in the cortex early in mammalian evolution as a result of these regulatory innovations. These results will provide fundamental insights into the molecular processes underlying the evolution of new morphological structures such as the neocortex. These results will also advance our understanding of human brain development, as they will provide information on the classes of genes and regulatory elements that may be particularly important for mammal- specific and cortex-specific functions in the brain.